Name: Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis (Video) | JoVE Journal
Uploaded: 2019-05-10T15:00:00.000+00:00
Duration: 10 min 31 s
Description: Learn the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying protein-bound RNA targets in mouse testis. This non-radioactive protocol improves signal specificity for studying spermatogenesis.

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We thank Eric L Van Nostrand and Gene W Yeo for helpful guidance with the original protocol. K.Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500), and National Natural Science Foundation of China (31771653). L.Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.

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<li>Goodier, J. L., Cheung, L. E., Kazazian, H. H. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MOV10+RNA+helicase+is+a+potent+inhibitor+of+retrotransposition+in+cells%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.</a> PLoS Genetics. 8 (10), e1002941(2012).</li>
<li>Haussecker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capped+small+RNAs+and+MOV10+in+human+hepatitis+delta+virus+replication%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">Capped small RNAs and MOV10 in human hepatitis delta virus replication.</a> Nature Structural & Molecular Biology. 15 (7), 714-721 (2008).</li>
<li>Kenny, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MOV10+and+FMRP+regulate+AGO2+association+with+microRNA+recognition+elements%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">MOV10 and FMRP regulate AGO2 association with microRNA recognition elements.</a> Cell Reports. 9 (5), 1729-1741 (2014).</li>
<li>Messaoudi-Aubert, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+for+the+MOV10+RNA+helicase+in+Polycomb-mediated+repression+of+the+INK4a+tumor+suppressor%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor.</a> Nature Structural & Molecular Biology. 17 (7), 862-868 (2010).</li>
<li>Sievers, C., Schlumpf, T., Sawarkar, R., Comoglio, F., Paro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mixture+models+and+wavelet+transforms+reveal+high+confidence+RNA-protein+interaction+sites+in+MOV10+PAR-CLIP+data%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data.</a> Nucleic Acids Research. 40 (20), e160(2012).</li>
<li>Skariah, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mov10+suppresses+retroelements+and+regulates+neuronal+development+and+function+in+the+developing+brain%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Mov10 suppresses retroelements and regulates neuronal development and function in the developing brain.</a> BMC Biology. 15 (1), 54(2017).</li>
<li>Zheng, K., Wang, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blockade+of+pachytene+piRNA+biogenesis+reveals+a+novel+requirement+for+maintaining+post-meiotic+germline+genome+integrity%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Blockade of pachytene piRNA biogenesis reveals a novel requirement for maintaining post-meiotic germline genome integrity.</a> PLoS Genetics. 8 (11), e1003038(2012).</li>
<li>Vourekas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+helicase+MOV10L1+binds+piRNA+precursors+to+initiate+piRNA+processing%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.</a> Genes & Development. 29 (6), 617-629 (2015).</li>
<li>Hocq, R., Paternina, J., Alasseur, Q., Genovesio, A., Le Hir, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitored+eCLIP%3A+high+accuracy+mapping+of+RNA-protein+interactions%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Monitored eCLIP: high accuracy mapping of RNA-protein interactions.</a> Nucleic Acids Research. 46 (21), 11553-11565 (2018).</li>
<li>Huppertz, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iCLIP%3A+protein-RNA+interactions+at+nucleotide+resolution%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">iCLIP: protein-RNA interactions at nucleotide resolution.</a> Methods. 65 (3), 274-287 (2014).</li>
<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome-wide+identification+of+RNA-binding+protein+and+microRNA+target+sites+by+PAR-CLIP%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.</a> Cell. 141 (1), 129-141 (2010).</li>
<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PAR-CliP--a+method+to+identify+transcriptome-wide+the+binding+sites+of+RNA+binding+proteins%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins.</a> Journal of Visualized Experiments. (41), (2010).</li>
<li>Konig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iCLIP+reveals+the+function+of+hnRNP+particles+in+splicing+at+individual+nucleotide+resolution%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.</a> Nature Structural & Molecular Biology. 17 (7), 909-915 (2010).</li>
<li>Konig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((iCLIP--transcriptome-wide+mapping+of+protein-RNA+interactions+with+individual+nucleotide+resolution%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution.</a> Journal of Visualized Experiments. (50), (2011).</li>
<li>Maticzka, D., Ilik, I. A., Aktas, T., Backofen, R., Akhtar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((uvCLAP+is+a+fast+and+non-radioactive+method+to+identify+in+vivo+targets+of+RNA-binding+proteins%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">uvCLAP is a fast and non-radioactive method to identify in vivo targets of RNA-binding proteins.</a> Nature Communications. 9 (1), 1142(2018).</li>
<li>Van Nostrand, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+transcriptome-wide+discovery+of+RNA-binding+protein+binding+sites+with+enhanced+CLIP+%28eCLIP%29%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).</a> Nature Methods. 13 (6), 508-514 (2016).</li>
<li>Radulovich, N., Leung, L., Tsao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modified+gateway+system+for+double+shRNA+expression+and+Cre%2Flox+based+gene+expression%5BTitle%5D)+AND+%22BMC+Biotechnology%22%5BJournal%5D)">Modified gateway system for double shRNA expression and Cre/lox based gene expression.</a> BMC Biotechnology. 11, 24(2011).</li>
<li>Breunig, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Customizable+Protocol+for+String+Assembly+gRNA+Cloning+%28STAgR%29%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A Customizable Protocol for String Assembly gRNA Cloning (STAgR).</a> Journal of Visualized Experiments. (142), (2018).</li>
<li>Kuhn, R. M., Haussler, D., Kent, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+UCSC+genome+browser+and+associated+tools%5BTitle%5D)+AND+%22Briefings+in+Bioinformatics%22%5BJournal%5D)">The UCSC genome browser and associated tools.</a> Briefings in Bioinformatics. 14 (2), 144-161 (2013).</li>
<li>Vourekas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mili+and+Miwi+target+RNA+repertoire+reveals+piRNA+biogenesis+and+function+of+Miwi+in+spermiogenesis%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis.</a> Nature Structural & Molecular Biology. 19 (8), 773-781 (2012).</li>
<li>Preitner, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((APC+is+an+RNA-binding+protein%2C+and+its+interactome+provides+a+link+to+neural+development+and+microtubule+assembly%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">APC is an RNA-binding protein, and its interactome provides a link to neural development and microtubule assembly.</a> Cell. 158 (2), 368-382 (2014).</li>
<li>Meyer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+TIA1+RNA-Binding+Protein+Family+Regulates+EIF2AK2-Mediated+Stress+Response+and+Cell+Cycle+Progression%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression.</a> Molecular Cell. 69 (4), 622-635 (2018).</li>
<li>Gerstberger, S., Hafner, M., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+census+of+human+RNA-binding+proteins%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">A census of human RNA-binding proteins.</a> Nature Reviews Genetics. 15 (12), 829-845 (2014).</li>
<li>Vourekas, A., Mourelatos, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HITS-CLIP+%28CLIP-Seq%29+for+mouse+Piwi+proteins%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">HITS-CLIP (CLIP-Seq) for mouse Piwi proteins.</a> Methods in Molecular Biology. 1093, 73-95 (2014).</li>
</ol>

The authors have nothing to disclose.

With increasing understanding of the universal role of RBPs under both biological and pathological contexts, the CLIP methods have been widely utilized to reveal the molecular function of RBPs20,32,33,34,35. The protocol described here represents an adapted application of the eCLIP method to mouse testis.
One challenge in performing...

Mammalian testis represents an excellent developmental model wherein an intricate cell differentiation program runs cyclically to yield numerous spermatozoa. An unique value of this model lies in the emergence of transcriptional inactivation at certain stages of spermatogenesis, typically when meiotic sex chromosome inactivation (MSCI) occurs1,2 and when round spermatids undergo drastic nuclear compaction during spermiogenesis3. These inconsecutive transcriptional events necessitate post-transcriptional gene regulation, in which RNA-binding proteins (RBPs) play a crucial role, shaping transcriptome and maintaining male fertility.
To identify the bona fide RNA targets of an individual RBP in vivo, the crosslinking immunoprecipitation (CLIP) method was developed4,5, based on but beyond the regular RNA immunoprecipitation (RIP)6,7, by incorporation of key steps including ultraviolet (UV) crosslinking, stringent wash and gel transfer to improve signal specificity. The advanced application of the CLIP combined with high-throughput sequencing has provoked large interest in profiling protein-RNA interaction at genome-wide levels8. In addition to genetic studies on RBP function, such biochemical methods that identify the direct interplay of endogenous protein and RNA have been indispensable to accurately elucidate the RNA regulatory roles of RBPs. For example, MOV10L1 is a testis-specific RNA helicase required for male fertility and the Piwi-interacting RNA (piRNA) biogenesis9. Its paralogue MOV10 is known as a ubiquitously expressed and multifunctional RNA helicase with roles in multiple aspects of RNA biology10,11,12,13,14,15,16,17,18. By employing the conventional CLIP-seq, we found that MOV10L1 binds and regulates primary piRNA precursors to initiate early piRNA processing19,20, and that MOV10 binds mRNA 3&#39; UTRs and as well as noncoding RNA species in testicular germ cells (data not shown).
Nevertheless, CLIP is originally a laborious, radioactive procedure followed by sequencing library preparation with a remarkable loss of CLIP tags. In the conventional CLIP, a cDNA library is prepared using adapters ligated at both RNA extremities. After protein digestion, crosslinked short polypeptides remain attached to RNA fragments. This crosslinking mark partially blocks reverse transcriptase (RTase) progression during cDNA synthesis, resulting in truncated cDNAs which represent about 80% of the cDNA library21,22. Thus, only cDNA fragments resulting from RTase bypassing the crosslinking site (read-through) are sequenced. Recently, various CLIP approaches, such as PAR-CLIP, iCLIP, eCLIP and uvCLAP, have been employed to identify crosslink sites of RBPs in living cells. PAR-CLIP involves the application of 365 nm UV radiation and photoactivatable nucleotide analogs and is therefore exclusive to in-culturing living cells, and incorporation of nucleoside analogs into newly synthesized transcripts is prone to producing bias where RNA physically interacts with protein23,24. In iCLIP, only a single adapter is ligated to the 3&#39; extremity of crosslinked RNA fragments. After reverse transcription (RT), both truncated and read-through cDNAs are obtained by intramolecularly circularization and re-linearization followed by polymerase chain reaction (PCR) amplification25,26. However, the efficiency of intramolecular circularization is relatively low. Although older CLIP protocols need labeling of crosslinked RNA with a radioisotope, ultraviolet crosslinking and affinity purification (uvCLAP), with a process of stringent tandem affinity purification, does not rely on radioactivity27. Nevertheless, uvCLAP is limited to cultured cells that must be transfected with the expression vector carrying the 3x FLAG-HBH tag for tandem affinity purification.
In eCLIP, adapters were ligated first at the 3&#39; extremity of RNA followed by RT, and next at the 3&#39; extremity of cDNAs in an intermolecular mode. Hence, eCLIP is able to capture all truncated and read-through cDNA28. Also, it is neither restricted to radioactive labeling, nor to using cell lines based on its principle, while maintaining single-nucleotide resolution.
Here, we provide a step-by-step description of an eCLIP protocol adapted for mouse testis. Briefly, this eCLIP protocol starts with UV crosslinking of testicular tubules, followed by partial RNase digestion and immunoprecipitation using a protein-specific antibody. Next, the protein-bound RNA is dephosphorylated, and adapter is ligated to its 3&#39; end.&#160;After protein gel electrophoresis and gel-to-membrane transfer, RNA is isolated by cutting the membrane area of an expected size range. After RT, DNA adapter is ligated to the 3&#39; end of cDNA followed by PCR amplification. Screening of subclones prior to high-throughput sequencing is taken as a library quality control. This protocol is efficient at identifying major species of protein-bound RNA of RBPs, exemplified by the two testis-expressing RNA helicases MOV10L1 and MOV10.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse MOV10&amp;nbsp; antibody</td><td>Proteintech, China</td><td>10370-1-AP</td><td></td></tr><tr><td>Anti-mouse MOV10L1 antibody</td><td>Zheng et al.2010&lt;sup&gt;9&lt;/sup&gt;</td><td>polyclonal antisera UP2175</td><td>provided by P. Jeremy Wang lab, University of Pennsylvania</td></tr><tr><td>HRP Goat Anti-Rabbit IgG&amp;nbsp;</td><td>ABclonal</td><td>AS014</td><td></td></tr><tr><td>Rabbit IgG</td><td>Beyotime, China</td><td>A7016</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hamburg, Germany</td><td>5242R</td><td></td></tr><tr><td>Digital sonifier</td><td>BRANSON,USA</td><td>BBV12081048A</td><td>450 Watts; 50/60 Hz</td></tr><tr><td>DynaMag-2 Magnet</td><td>Invitrogen,USA</td><td>12321D</td><td></td></tr><tr><td>Mini Blot Module</td><td>Invitrogen,USA</td><td>B1000</td><td></td></tr><tr><td>Mini Gel Tank</td><td>Invitrogen,USA</td><td>A25977</td><td></td></tr><tr><td>Shaking incubator</td><td>Eppendorf, Hamburg, Germany</td><td>Thermomixer comfort&amp;nbsp;</td><td></td></tr><tr><td>Tissue Grinder, Dounce</td><td>PYREX, USA</td><td>1234F35</td><td>only&amp;nbsp; the &quot;loose&quot; pestle is used in this protocol</td></tr><tr><td>TProfessional standard 96 Gradient</td><td>Biometra, Germany</td><td>serial no.: 2604323</td><td></td></tr><tr><td>Tube Revolver&amp;nbsp;</td><td>Crystal, USA</td><td>serial no.: 3406051</td><td></td></tr><tr><td>UV-light cross-linker</td><td>UVP, USA</td><td>CL-1000</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430167</td><td>100 mm&amp;nbsp;</td></tr><tr><td>Tubes</td><td>Corning, USA</td><td>430791</td><td>15 mL</td></tr><tr><td>Microtubes tubes</td><td>AXYGEN , USA</td><td>MCT-150-C</td><td>1.5 mL&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acid phenol/chloroform/isoamyl alcohol&amp;nbsp;</td><td>Solarbio, China</td><td>P1011</td><td>25:24:01</td></tr><tr><td>AffinityScript Enzyme</td><td>Agilent, USA</td><td>600107</td><td></td></tr><tr><td>Antioxidant</td><td>Invitrogen,USA</td><td>NP0005</td><td></td></tr><tr><td>DH5&amp;alpha; competent bacteria</td><td>Thermo Scientific, USA</td><td>18265017</td><td>these economical cells yield &gt;1 x 10&lt;sup&gt;6&lt;/sup&gt; transformants/&amp;micro;g control DNA per 50 &amp;micro;L reaction.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich, USA</td><td>D8418</td><td></td></tr><tr><td>DNA Ladder</td><td>Invitrogen, USA</td><td>10416014</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich, USA</td><td>DNTP100-1KT</td><td></td></tr><tr><td>Dynabeads Protein A&amp;nbsp;</td><td>Invitrogen, USA</td><td>10002D</td><td></td></tr><tr><td>ECL reagent</td><td>Vazyme, China</td><td>E411-04</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen, USA</td><td>AM9260G</td><td></td></tr><tr><td>EDTA free protease inhibitor cocktail</td><td>Roche, USA</td><td>04693132001</td><td>add fresh</td></tr><tr><td>Exo-SAP-IT</td><td>Affymetrix, USA</td><td>78201</td><td>PCR Product Cleanup Reagent&amp;nbsp;</td></tr><tr><td>FastAP enzyme</td><td>Thermo Scientific, USA</td><td>EF0652</td><td></td></tr><tr><td>LDS Sample Buffer</td><td>Thermo Scientific, USA</td><td>NP0007</td><td></td></tr><tr><td>MetaPhor Agarose&amp;nbsp;</td><td>lonza, Switzerland</td><td>50180</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Invitrogen, USA</td><td>AM9530G</td><td></td></tr><tr><td>MiniElute gel Extraction</td><td>QIAGEN, Germany</td><td>28604</td><td>column store at 4 ?; buffer QG=gel dissolving buffer; buffer PE= wash buffer(for step 14)</td></tr><tr><td>MyONE Silane beads</td><td>Thermo Scientific, USA</td><td>37002D</td><td>nucleic acids extraction magnetic beads</td></tr><tr><td>NaCl</td><td>Invitrogen,USA</td><td>AM9759&lt;br/&gt; &amp;nbsp;</td><td></td></tr><tr><td>NP-40</td><td>Amresco, USA</td><td>M158-500ML</td><td></td></tr><tr><td>NuPAGE Bis-Tris Protein Gels</td><td>Invitrogen, USA</td><td>NP0336BOX</td><td>4%&amp;ndash;12%,1.5 mm, 15-well</td></tr><tr><td>NuPAGE MOPS SDS Buffer Kit&amp;nbsp;</td><td>Invitrogen, USA</td><td>NP0050</td><td></td></tr><tr><td>PBS</td><td>Gibco, USA</td><td>10010023</td><td></td></tr><tr><td>Phase-Locked Gel (PLG) heavy tube&amp;nbsp;&amp;nbsp;</td><td>TIANGEN, China</td><td>WM5-2302831</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>Applied Biosystems, USA</td><td>A25742</td><td></td></tr><tr><td>Proteinase K</td><td>NEB, New England&amp;nbsp;</td><td>P8107S</td><td></td></tr><tr><td>Q5 PCR master mix&amp;nbsp;</td><td>NEB, New England&amp;nbsp;</td><td>M0492L</td><td></td></tr><tr><td>RLT buffer</td><td>QIAGEN, Germany</td><td>79216</td><td>RNA purification lysis buffer&amp;nbsp;</td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5 columns&amp;nbsp;</td><td>ZYMO RESEARCH, USA</td><td>R1016</td><td>RNA purification and concentration columns</td></tr><tr><td>RNase I&amp;nbsp;</td><td>Invitrogen, USA</td><td>AM2295</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Promega, USA</td><td>N251B</td><td></td></tr><tr><td>RQ1 DNase</td><td>Promega,USA</td><td>M610A</td><td></td></tr><tr><td>Sample Reducing Agent</td><td>Invitrogen,USA</td><td>NP0009</td><td></td></tr><tr><td>SDS Solution</td><td>Invitrogen, USA</td><td>15553027</td><td>10%</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich, USA</td><td>30970</td><td>protect from light</td></tr><tr><td>T4 PNK enzyme</td><td>NEB, New England&amp;nbsp;</td><td>M0201L</td><td></td></tr><tr><td>T4 RNA ligase 1 high conc</td><td>NEB, New England&amp;nbsp;</td><td>M0437M</td><td></td></tr><tr><td>TA/Blunt-Zero Cloning Mix</td><td>Vazyme, China</td><td>C601-01</td><td></td></tr><tr><td>TBE</td><td>Invitrogen,USA</td><td>AM9863</td><td></td></tr><tr><td>Tris-HCI Buffer</td><td>Invitrogen, USA</td><td>15567027</td><td></td></tr><tr><td>Triton X-100</td><td>Sangon Biotech, China</td><td>A600198&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sangon Biotech, China</td><td>A600560</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich, USA</td><td>U5378</td><td></td></tr><tr><td>X-ray Films</td><td>Caresteam, Canada</td><td>6535876</td><td></td></tr></tbody></table>

enhanced crosslinking immunoprecipitation (eclip) method for efficient identification of protein-bound rna in mouse testis

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. This study establishes an applicable basis for eCLIP in mammalian testis.

The eCLIP procedure and results are illustrated in Figure 1, Figure 2, Figure 3, Figure 4. Mice were euthanized with carbon dioxide and a small incision was made in the lower abdomen using surgical scissors (Figure 2A,B). Mo...

Here, we present an eCLIP protocol to determine major RNA targets of RBP candidates in testis.

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, ...

enhanced-crosslinking-immunoprecipitation-eclip-method-for-efficient

Research

JoVE Journal

Biology

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

This video demonstrates the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying RNA-binding proteins in mouse testis. eCLIP enables the precise identification of binding sites where RNA-binding proteins (RBPs) interact with their target RNAs.

An Enhanced Crosslinking Immunoprecipitation Method for Efficient Identification of Protein-bound RNA in Mouse Testes

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

A UV-based ChIRP method to identify direct lncRNA-protein interactions.

A UV-Based ChIRP Method For The Verifiable Identification of Proteins That Directly Interact With LncRNA: From Cultured Cells To Mouse Testis

Learn the enhanced crosslinking immunoprecipitation (eCLIP) method for identifying protein-bound RNA targets in mouse testis. This non-radioactive protocol improves signal specificity for studying spermatogenesis.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

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